Project Abstract: Menopause increases the risk and severity of ischemic stroke and increases the subsequent risk for vascular dementia. Clinical and experimental studies indicate that the loss of estrogen contributes to elevated risk for both diseases. However, the landmark Women?s Health Initiative study, showed that hormone therapy increased stroke and dementia. Our studies have shown that ischemic stroke outcomes are worse in middle- aged, reproductive senescent (acyclic) female rats, as compared to younger normally cycling female rats. Moreover, estrogen replacement to the acyclic group exacerbates stroke outcomes. In the previous funding period, we identified epigenetic changes in the aging astrocyte as a key factor in poor stroke recovery in reproductive senescent females. Compared to adult females, astrocytes from the ischemic cortex of middle-aged female rats show a reduced ability to sequester glutamate, decreased growth factor release and increased release of chemokines, suggesting that the neuroprotective capacity of astrocytes is reduced with reproductive aging. Our studies further showed that astrocytes from middle-aged females exhibit deleterious epigenetic modifications in histone methylation and miRNA, that exacerbate behavioral and anatomical effects of stroke. This renewal is based on our observation that microRNA (mir)20a-3p is expressed at similar levels in adult and middle-aged female astrocytes prior to stroke, but shows a remarkable age-specific regulation in the acute phase of stroke. Thus, mir20a-3p is elevated in adult female astrocytes, but profoundly downregulated in middle-aged astrocytes. Mir20a-3p is predicted to repress mitochondrial genes and inflammatory mediators, and consequently, its loss in estrogen-deficient middle-aged females may preserve mitochondrial function in astrocytes in the acute injury period, but also result in increased inflammation and blood brain barrier disruption, and ultimately, poor recovery. This potential for opposing functions is the basis for our hypothesis that mir20a-3p reduction in the reproductive senescent astrocyte will promote cell survival in the acute ischemic period, but in the long term, elevating mir20-3p levels will result in increased protection against impairment due to increased infarction. This hypothesis will be tested in 3 aims: Aim 1) mir20a-3p/5p suppression is an astrocyte-specific response to stroke in middle-aged female rats, Aim 2) temporal manipulation of astrocytic mir20a-3p is neuroprotective and anti-inflammatory for ischemic stroke and Aim 3) mir20a-3p treatment reduces long term degeneration in cognitive-affective circuits associated with vascular dementia. Using robust and clinically relevant stroke models, multiple outcome measures and modern gene transfer tools, this application will critically evaluate the extent to which a) some aspects of aging are compensatory and neuroprotective, and b) these compensations may be ?pharmacological? manipulated in astrocytes as a novel and unstudied target for neurologic diseases that emerge after reproductive senescence.